The long term goal of this proposal is the elucidation of the molecular mechanisms by which the assembly of microtubules is regulated. Specifically, I wish to investigate the following three questions. First, what is the genetic complexity of microtubule proteins? On a genetic level, how many tubulin genes are there and what is the chromosomal organization of these genes? Are different tubulin genes differentially expressed during development, during differentiation, or as a function of the cell cycle? The methodology involved in answering these questions centers on the use of cDNA and genomic clones encoding Alpha or Beta tubulins. I have already constructed and characterized the requisite cDNA clones from embryonic chick brain mRNA. Many phage isolates carrying genomic chick tubulin sequences have also been screened from phage libraries. Second, how is the level of synthesis of Alpha and Beta tubulin controlled? Experiments in two laboratories have suggested that level of unpolymerized tubulin present in a cell modulates the level of new tubulin synthesis by altering the available levels of translatable tubulin mRNAs. This phenomonon may function as a sensitive control element of table tubulin mRNAs. This phenomonon may function as a sensitive control element of tubulin polyermization. Is the biochemical mechanism for such control exercised at the level of transcription, processing, degredation, and/or translation of the tubulin mRNA? Moreover, are all tubulin mRNAs subject to this regulation and is the synthesis of other microtubule proteins controlled in a coordinate manner. Again methodologies for the assay of tubulin mRNA transcription, etc., will rely on the use of cloned copies of tubulin nucleic acid sequences. Third, how do microtubule associated proteins participate in microtubule assembly or function? In particular, what is the source of heterogeneity of tau, a microtubule associated protein originally identified by its ability to stimulate tubulin polymerization? What are the initial translation product(s) of tau, is heterogeneity of tau correlated with tubulin polymerization in vivo, and is tau specifically modified in a developmental or cell cycle dependent manner? For these studies, monospecific antibodies suitable for use in immunoprecipitation of tau through developmental programs or during differentiation events have been prepared.